It has been known that fruit juice raw materials to be processed into fruit juice drinks and fruit juice processed food products may contain thermotolerant acidophilic bacteria (TAB) which are viable in conventional acidic drinks even under heat sterilization conditions of food products. Among typical thermotolerant acidophilic bacteria belonging to the genus Alicyclobacillus, Alicyclobacillus acidoterrestris (AAT) and Alicyclobacillus acidiphilus (AAP) have been known as the bacteria that produce guaiacol (hereinafter referred to as guaiacol-producing bacteria). Particularly, AAT bacteria have been reported to produce guaiacol via vanillin and vanillic acid (Japan Fruit Juice Association Newsletter (KAJU KYOKAI HO), December 2005, p. 1-15; Non-Patent Document 2). Guaiacol is not toxic, but has an unusual odor. Thus, products such as drinks containing guaiacol-producing bacteria have significantly deteriorated flavor. In many cases, AAT in fruit juice raw materials was identified to be a primary cause. Therefore, it is very important to assay the presence of guaiacol-producing bacteria in fruit juice raw materials and fruit juice drink products for quality controls of raw materials and products.
It has been known that AAT can be detected by a method comprising adding a fruit juice drink to a liquid medium containing vanillin, incubating the mixture and measuring the odor of guaiacol by a sensory test (Japanese Patent Application Laid-open No. H7-123998; Patent Document 1); or by a method for detecting the presence of guaiacol-producing bacteria comprising incubating a solution containing a specimen in the presence of vanillic acid and treating the produced guaiacol with peroxidase to develop a color followed by a qualitative or spectrometric quantitative analysis using a particular equipment (Japanese Patent Application Laid-open No. 2004-201668; Patent Document 2); or by PCR detection using primers specific to the gene of a guaiacol producing enzyme (Japanese Patent Application Laid-open No. 2003-000259; Patent Document 3).
The technique based on the above Patent Document 2 is now widely used for AAT detection with an indirect assay for the presence of AAT comprising culturing a specimen in a liquid medium containing vanillic acid and detecting the produced guaiacol with a peroxidase method.
However, the peroxidase method accompanies complicated procedures and requires a prolonged time before obtaining results because an adequate preculture step is required to grow AAT in a liquid medium. For example, in order to quantitatively detect AAT in a specimen, it is necessary to form colonies on a plate medium, pick more than one colony and culture respective colonies in a liquid medium, which are complicated procedures and require a period of 4 to 6 days.
Moreover, in the peroxidase method, the presence or absence of AAT is determined by brownish coloration produced by transformation of guaiacol to tetraguaiacol by the action of peroxidase; thus, determination may be difficult when a specimen is a raw material or medium having similar color such as orange juice. A certain skill is required for personnel who perform visual colorimetric determinations, because the color is influenced by different lots of raw materials and from conditions for autoclaving the media. For accurate determinations, spectrophotometric analysis with calibration curves for each specimen and medium, or GC-MS analysis are required, and thus expensive analyzers and complex procedures are further required.
Another known method using a solid medium is a 30° C.-culture method, where guaiacol-producing bacteria are distinguished from guaiacol non-producing bacteria based on culture temperatures. For example, a specimen is cultured at 30° C., which is lower than an optimal growth temperature of AAT (40 to 50° C.), and the presence or absence of colonies is determined. Because the growth of AAT is also decreased in culture at 30° C., the sensitivity of detection of AAT is low and guaiacol non-producing bacteria of no interest having a lower optimal growth temperature range will also emerge. Accordingly, a preculture step is required for the 30° C.-culture method in order to improve the sensitivity of detection of AAT. In this case, all the steps may take as long time as 8 to 10 days, and the selectivity is not sufficient. Moreover the method is not quantitative because the preculture step is required.
In addition, it has been known that components such as polyphenols contained in citrus fruits and grapes may inhibit the growth of AAT. Thus, a specimen needs to be appropriately diluted before an assay of raw materials having high fruit concentrations such as 100% fruit juices. For example, orange juice is diluted to 1% before an assay. However, the dilution factor needs to be reviewed per type of fruit juice and the production area thereof, making the above assay procedures more complicated.
Therefore, there still exist a need for a quick and accurate method for determination of the presence or absence of guaiacol-producing bacteria in fruit juice raw materials without requiring complicated procedures, expensive equipments or a skill of operators. There is also a need for a convenient method which allows assays at the sites of production of fruits and in facilities for production of fruit juice raw material.
The references cited in the present application are as follows. The contents thereof are entirely incorporated herein by reference.
Patent Document 1: Japanese Patent Application Laid-open No. H7-123998
Patent Document 2: Japanese Patent Application Laid-open No. 2004-201668
Patent Document 3: Japanese Patent Application Laid-open No. 2003-000259
Non-Patent Document 1: “The Unified Test Method for Thermo-Acidophilic Bacilli”, Japan Fruit Juice Association, 2005, p. 25
Non-Patent Document 2: Japan Fruit Juice Association Newsletter (KAJU KYOKAI HO), December 2005, p. 1-15